406
Appendix
Cp medium (Miltenburger et aI., 1984)
The concentrations are given as mgll,OOO ml medium.
KCI 2,870.0, CaCI 2 ·2H 2 0* 1,320.0, MgCI 2 ·6H 2 0* 2,280.0, MgS0 4 ·7H 2 0* 2,780.0,
NaH 2 P0 4 ·2H 2 0* 1,140.0, NaHC03 350.0, L-arginine hydrochloride 350.0, L-aspartic acid
175.0, L-asparagine 175.0, L-alanine 112.5, ~-alanine 100.0, L-cystine 11.0, L-glutamic
acid 300.0, L-glutamine 300.0, glycine 320.0, L-histidine hydrochloride 1,250.0, L-isoleucine 25.0, L-Ieucine 37.5, L-Iysine hydrochloride 312.5, L-methionine 25.0g, L-proline 175.0, L-phenylalanine 75.0, DL-serine 550.0, L-threonine 87.5, L-tryptophan 50.0,
L-tyrosine 25.0, L-valine 50.0, p-aminobenzoic acid 0.010, biotin 0.005, D-calcium pantothenate 0.010, choline chloride 0.100, folic acid 0.010, i-inositol 0.010, nicotinic acid
0.010, pyridoxine hydrochloride 0.010, riboflavin 0.010, thiamine hydrochloride 0.010,
glucose 1,000.0, and tryptose 2,600.0, made up to 1,000 ml with distilled water. The
medium is supplemented with 10% FBS. The pH is adjusted to 6.4 with 1 N KOH.
Crab integument medium I and II (Ballard et aI., 1993)
Stock solutions:
Modified medium 199 (MI99). Dissolve 10,900 mg Medium 199 (Sigma) in 900 ml
deionized-distilled water (DDW).Add 5,960 mg HEPES,200 mg MgCI2·6H 2 0, 15,000 mg
NaCI, and 200 mg L-proline and adjust the pH to 7.5 with 1 N NaOH or 1 N HCI. Make
the solution up to a final volume of 1,000 ml with DDW. Sterilize with a 0.22-l1m filter
and store at 4°C.
Antibiotic cocktails (HCA). In 900 ml DDW, dissolve 100 mg Benomyl (Dragon Chemical), 50 mg gentamicin sulfate, 25 mg nystatin, 100 mg penicillin G. When in solution,
add 1.65 ml penicillin-streptomycin solution (Sigma), 0.5 ml gentamicin sulfate solution (Sigma), and 0.5 ml fungizone (Gibco).Adjust the pH to 7.5 with 1 N NaOH or 1 N
Hel. Make the solution up to a final volume of 1,000 ml with DDW. Sterilize through a
0.22-l1m filter and store in 7.65 ml aliquots at -20°C (thaw before use in final medium).
Antibiotic cocktails (LCA). Sterilize the penicillin-streptomycin solution (Sigma), gentamicin sulfate solution (Sigma), and fungizone (Gibco) by passage through a 0.22-l1m
filter. Into separate sterile tubes, aliquot 1.65 ml penicillin-streptomycin, 0.5 ml gentamicin sulfate and 0.5 ml fungizone. Store the aliquots at -20°C (thaw before use in
final medium).
Complete medium I: mix 95 ml M119 and 5 ml FBS. Take 7.65 ml this mixture and add
a 7.65 ml HCA aliquot. Sterilize the mixture by passage through a 0.22-l1m filter and
store the solution at 4°C (warm to room temperature before use).
Complete medium II: mix 95 ml M119 and 5 ml FBS. Take 2.65 ml this mixture and
add one aliquot of LCA. Sterilize the solution by passage through a 0.22-l1m filter and
store at 4°C (warm to room temperature before use).
Crayfish integument medium (Daig and Spindler, 1979)
Mix 2 parts of van Harrevelt saline with 1 part horse serum; osmolarity 349 mOsmoll
kg, pH 7.6.
CSM-2F medium (Mitsuhashi, 1967)
Unless otherwise indicated, the concentrations are given as mg/1,000 ml medium.
NaH 2 P0 4 ·Hp 500, MgCI 2 ·6H 2 0 1,200, MgS0 4 ·7H 2 0 1,600, KCl1,200, CaCI 2 ·2H 2 0 400,
glucose 800, fructose 800, lactalbumin hydrolysate 5,200, Bacto-peptone (Difco Laboratories) 5,200, choline chloride 400, TC-Yeas to late 2,000, TC-199 medium 200 ml, FBS
Appendix
Cp medium (Miltenburger et aI., 1984)
The concentrations are given as mgll,OOO ml medium.
KCI 2,870.0, CaCI 2 ·2H 2 0* 1,320.0, MgCI 2 ·6H 2 0* 2,280.0, MgS0 4 ·7H 2 0* 2,780.0,
NaH 2 P0 4 ·2H 2 0* 1,140.0, NaHC03 350.0, L-arginine hydrochloride 350.0, L-aspartic acid
175.0, L-asparagine 175.0, L-alanine 112.5, ~-alanine 100.0, L-cystine 11.0, L-glutamic
acid 300.0, L-glutamine 300.0, glycine 320.0, L-histidine hydrochloride 1,250.0, L-isoleucine 25.0, L-Ieucine 37.5, L-Iysine hydrochloride 312.5, L-methionine 25.0g, L-proline 175.0, L-phenylalanine 75.0, DL-serine 550.0, L-threonine 87.5, L-tryptophan 50.0,
L-tyrosine 25.0, L-valine 50.0, p-aminobenzoic acid 0.010, biotin 0.005, D-calcium pantothenate 0.010, choline chloride 0.100, folic acid 0.010, i-inositol 0.010, nicotinic acid
0.010, pyridoxine hydrochloride 0.010, riboflavin 0.010, thiamine hydrochloride 0.010,
glucose 1,000.0, and tryptose 2,600.0, made up to 1,000 ml with distilled water. The
medium is supplemented with 10% FBS. The pH is adjusted to 6.4 with 1 N KOH.
Crab integument medium I and II (Ballard et aI., 1993)
Stock solutions:
Modified medium 199 (MI99). Dissolve 10,900 mg Medium 199 (Sigma) in 900 ml
deionized-distilled water (DDW).Add 5,960 mg HEPES,200 mg MgCI2·6H 2 0, 15,000 mg
NaCI, and 200 mg L-proline and adjust the pH to 7.5 with 1 N NaOH or 1 N HCI. Make
the solution up to a final volume of 1,000 ml with DDW. Sterilize with a 0.22-l1m filter
and store at 4°C.
Antibiotic cocktails (HCA). In 900 ml DDW, dissolve 100 mg Benomyl (Dragon Chemical), 50 mg gentamicin sulfate, 25 mg nystatin, 100 mg penicillin G. When in solution,
add 1.65 ml penicillin-streptomycin solution (Sigma), 0.5 ml gentamicin sulfate solution (Sigma), and 0.5 ml fungizone (Gibco).Adjust the pH to 7.5 with 1 N NaOH or 1 N
Hel. Make the solution up to a final volume of 1,000 ml with DDW. Sterilize through a
0.22-l1m filter and store in 7.65 ml aliquots at -20°C (thaw before use in final medium).
Antibiotic cocktails (LCA). Sterilize the penicillin-streptomycin solution (Sigma), gentamicin sulfate solution (Sigma), and fungizone (Gibco) by passage through a 0.22-l1m
filter. Into separate sterile tubes, aliquot 1.65 ml penicillin-streptomycin, 0.5 ml gentamicin sulfate and 0.5 ml fungizone. Store the aliquots at -20°C (thaw before use in
final medium).
Complete medium I: mix 95 ml M119 and 5 ml FBS. Take 7.65 ml this mixture and add
a 7.65 ml HCA aliquot. Sterilize the mixture by passage through a 0.22-l1m filter and
store the solution at 4°C (warm to room temperature before use).
Complete medium II: mix 95 ml M119 and 5 ml FBS. Take 2.65 ml this mixture and
add one aliquot of LCA. Sterilize the solution by passage through a 0.22-l1m filter and
store at 4°C (warm to room temperature before use).
Crayfish integument medium (Daig and Spindler, 1979)
Mix 2 parts of van Harrevelt saline with 1 part horse serum; osmolarity 349 mOsmoll
kg, pH 7.6.
CSM-2F medium (Mitsuhashi, 1967)
Unless otherwise indicated, the concentrations are given as mg/1,000 ml medium.
NaH 2 P0 4 ·Hp 500, MgCI 2 ·6H 2 0 1,200, MgS0 4 ·7H 2 0 1,600, KCl1,200, CaCI 2 ·2H 2 0 400,
glucose 800, fructose 800, lactalbumin hydrolysate 5,200, Bacto-peptone (Difco Laboratories) 5,200, choline chloride 400, TC-Yeas to late 2,000, TC-199 medium 200 ml, FBS